Wandong Zhang

The expression and functional characterization of ABCG2 in brain endothelial cells and vessels.

Delivery of drugs to the brain is impeded by the activity of efflux pumps expressed by endothelial cells of brain vasculature. The ATP binding cassette (ABC) transporters, among which ABCB1/MDR1 P‐glycoprotein and ABCC1/multidrug resistance‐associated protein 1 are expressed in brain endothelial cells, participate in drug efflux properties of the blood‐brain barrier (BBB). Searches of the EST (expressed sequence tags) database with the conserved ABC domain, conducted to identify other ABC transporters expressed in the BBB, recovered 15 ABC transporter sequences expressed in human brain cDNA libraries. One of these sequences, identical to ABCG2, was highly expressed in cultured human cerebromicrovascular endothelial cells and human brain tissue at both mRNA and protein levels. Overexpression of human ABCG2 in immortalized rat brain endothelial cells resulted in enhanced polarized abluminal to luminal transport of various substrates tested in the in vitro BBB model. Brain vessels extracted from tissue sections of nonmalignant human brain and glioblastoma tomors by laser capture microdissection microscopy and analyzed by real‐time polymerase chain reaction showed higher expression of ABCG2 relative to ABCB1/MDR1 and ABCC1/MRP1. ABCG2 was up‐regulated in both glioblastoma vessels and parenchymal tissue. These studies suggest a role for brain endothelial ABCG2 transporter in modulating drug delivery to the brain and in conferring drug resistance to glioblastomas.

ABCG2 Is Upregulated in Alzheimer's Brain with Cerebral Amyloid Angiopathy and May Act as a Gatekeeper at the Blood–Brain Barrier for Aβ1–40 Peptides

Alzheimers disease (AD) is characterized by accumulation and deposition of Aβ peptides in the brain. Aβ deposition in cerebrovessels occurs in many AD patients and results in cerebral amyloid angiopathy (AD/CAA). Since Aβ can be transported across blood–brain barrier (BBB), aberrant Aβ trafficking across BBB may contribute to Aβ accumulation in the brain and CAA development. Expression analyses of 273 BBB-related genes performed in this study showed that the drug transporter, ABCG2, was significantly upregulated in the brains of AD/CAA compared with age-matched controls. Increased ABCG2 expression was confirmed by Q-PCR, Western blot, and immunohistochemistry. Abcg2 was also increased in mouse AD models, Tg-SwDI and 3XTg. Aβ alone or in combination with hypoxia/ischemia failed to stimulate ABCG2 expression in BBB endothelial cells; however, conditioned media from Aβ-activated microglia strongly induced ABCG2 expression. ABCG2 protein in AD/CAA brains interacted and coimmunoprecipitated with Aβ. Overexpression of hABCG2 reduced drug uptake in cells; however, interaction of Aβ1–40 with ABCG2 impaired ABCG2-mediated drug efflux. The role of Abcg2 in Aβ transport at the BBB was investigated in Abcg2-null and wild-type mice after intravenous injection of Cy5.5-labeled Aβ1–40 or scrambled Aβ40–1. Optical imaging analyses of live animals and their brains showed that Abcg2-null mice accumulated significantly more Aβ in their brains than wild-type mice. The finding was confirmed by immunohistochemistry. These results suggest that ABCG2 may act as a gatekeeper at the BBB to prevent blood Aβ from entering into brain. ABCG2 upregulation may serve as a biomarker of CAA vascular pathology in AD patients.

Expression of inflammatory genes induced by beta-amyloid peptides in human brain endothelial cells and in Alzheimer's brain is mediated by the JNK-AP1 signaling pathway

Alzheimers disease (AD) is characterized by accumulation and deposition of Abeta peptides in the brain. Abeta deposition in cerebral vessels occurs in many AD patients and results in cerebral amyloid angiopathy (AD/CAA). Abeta deposits evoke neuro- and neurovascular inflammation contributing to neurodegeneration. In this study, we found that exposure of cultured human brain endothelial cells (HBEC) to Abeta(1-40) elicited expression of inflammatory genes MCP-1, GRO, IL-1beta and IL-6. Up-regulation of these genes was confirmed in AD and AD/CAA brains by qRT-PCR. Profiling of 54 transcription factors indicated that AP-1 was strongly activated not only in Abeta-treated HBEC but also in AD and AD/CAA brains. AP-1 complex in nuclear extracts from Abeta-treated HBEC bound to AP-1 DNA-binding sequence and activated the reporter gene of a luciferase vector carrying AP-1-binding site from human MCP-1 gene. AP-1 is a dimeric protein complex and supershift assay identified c-Jun as a component of the activated AP-1 complex. Western blot analyses showed that c-Jun was activated via JNK-mediated phosphorylation, suggesting that as a result of c-Jun phosphorylation, AP-1 was activated and thus up-regulated MCP-1 expression. A JNK inhibitor SP600125 strongly inhibited Abeta-induced c-Jun phosphorylation, AP-1 activation, AP-1 reporter gene activity and MCP-1 expression in cells stimulated with Abeta peptides. The results suggested that JNK-AP1 signaling pathway is responsible for Abeta-induced neuroinflammation in HBEC and Alzheimers brain and that this signaling pathway may serve as a therapeutic target for relieving Abeta-induced inflammation.

Characterization of ABCB9, an ATP Binding Cassette Protein Associated with Lysosomes*

We have cloned full-length human and mouse cDNAs of ABCB9, which encodes a predicted multiple-spanning transmembrane domain and a nucleotide-binding domain with Walker motifs. It is therefore designated as a “half” ATP binding cassette (ABC) transporter. Northern analysis shows that the ABCB9 mRNA is expressed at a high level in testes and moderate levels in brain and spinal cord. A splice variant mRNA deleted in the last pair of predicted transmembrane segments was shown to be expressed in human tissues. Phylogenetic analysis indicates that ABCB9 is closely related to TAP1 and TAP2, two “half” ABC proteins found in endoplasmic reticulum. ABCB9 protein colocalized with the lysosomal markers, LAMP1 and LAMP2, in transfected cells. ABCB9 protein appears to be most highly expressed in the Sertoli cells of the seminiferous tubules in mouse and rat testes. These cells have high levels of phagocytosis and secretory activities. These findings pave the way for further investigation into the potential novel function of ABCB9 in lysosomes.

Hypoxia-inducible factor-1 (HIF-1) is involved in the regulation of hypoxia-stimulated expression of monocyte chemoattractant protein-1 (MCP-1/CCL2) and MCP-5 (Ccl12) in astrocytes

BackgroundNeuroinflammation has been implicated in various brain pathologies characterized by hypoxia and ischemia. Astroglia play an important role in the initiation and propagation of hypoxia/ischemia-induced inflammation by secreting inflammatory chemokines that attract neutrophils and monocytes into the brain. However, triggers of chemokine up-regulation by hypoxia/ischemia in these cells are poorly understood. Hypoxia-inducible factor-1 (HIF-1) is a dimeric transcriptional factor consisting of HIF-1α and HIF-1β subunits. HIF-1 binds to HIF-1-binding sites in the target genes and activates their transcription. We have recently shown that hypoxia-induced expression of IL-1β in astrocytes is mediated by HIF-1α. In this study, we demonstrate the role of HIF-1α in hypoxia-induced up-regulation of inflammatory chemokines, human monocyte chemoattractant protein-1 (MCP-1/CCL2) and mouse MCP-5 (Ccl12), in human and mouse astrocytes, respectively.MethodsPrimary fetal human astrocytes or mouse astrocytes generated from HIF-1α+/+ and HIF-1α+/- mice were subjected to hypoxia (<2% oxygen) or 125 μM CoCl2 for 4 h and 6 h, respectively. The expression of HIF-1α, MCP-1 and MCP-5 was determined by semi-quantitative RT-PCR, western blot or ELISA. The interaction of HIF-1α with a HIF-1-binding DNA sequence was examined by EMSA and supershift assay. HIF-1-binding sequence in the promoter of MCP-1 gene was cloned and transcriptional activation of MCP-1 by HIF-1α was analyzed by reporter gene assay.ResultsSequence analyses identified HIF-1-binding sites in the promoters of MCP-1 and MCP-5 genes. Both hypoxia and HIF-1α inducer, CoCl2, strongly up-regulated HIF-1α expression in astrocytes. Mouse HIF-1α+/- astrocytes had lower basal levels of HIF-1α and MCP-5 expression. The up-regulation of MCP-5 by hypoxia or CoCl2 in HIF-1α+/+ and HIF-1α+/- astrocytes was correlated with the levels of HIF-1α in cells. Both hypoxia and CoCl2 also up-regulated HIF-1α and MCP-1 expression in human astrocytes. EMSA assay demonstrated that HIF-1 activated by either hypoxia or CoCl2 binds to wild-type HIF-1-binding DNA sequence, but not the mutant sequence. Furthermore, reporter gene assay demonstrated that hypoxia markedly activated MCP-1 transcription but not the mutated MCP-1 promoter in transfected astrocytes.ConclusionThese findings suggest that both MCP-1 and MCP-5 are HIF-1 target genes and that HIF-1α is involved in transcriptional induction of these two chemokines in astrocytes by hypoxia.

Inflammatory Activation of Human Brain Endothelial Cells by Hypoxic Astrocytes In Vitro is Mediated by IL-1β

Leukocyte infiltration into the brain contributes to the development of ischemic brain damage and is mediated by endothelial/leukocyte adhesion molecules, cytokines, and chemokines released by ischemic brain cells. In this study, we provide evidence that human astrocytes (FHAs) subjected to in vitro hypoxia produce proinflammatory mediator(s) capable of up-regulating inflammatory genes, including intercellular adhesion molecule-1, interleukin (IL)-1β, tumor necrosis factor-α, IL-8, and monocyte chemotactic protein-1 (MCP-1) in human cerebromicrovascular endothelial cells (HCECs). FHAS were exposed to hypoxia in an anaerobic chamber for 4 hours, followed by reoxygenation for 24 hours. Astrocyte-conditioned media (ACM) collected from normoxic FHAS or FHAS subjected to hypoxia/reoxygenation were applied to HCEC cultures for 4 to 24 hours. Semiquantitative reverse transcription–polymerase chain reaction, immunocytochemistry, and enzyme-linked immunosorbent assay demonstrated up-regulation of intercellular adhesion molecule-1 in HCECs exposed to hypoxic ACM. A pronounced elevation in cytokine IL-1β and tumor necrosis factor-α, and chemokine IL-8 and MCP-1 mRNA, accompanied by increased release of immunoreactive cytokines and chemokines into cell media was observed in HCECs exposed to hypoxic ACM. Hypoxia/reoxygenation induced a transient (4 to 18 hours of reoxygenation) upregulation of IL-1β mRNA in FHAS and a two- to threefold increase in IL-1β levels secreted into ACM. Pretreatment of FHAS with 10 μmol/L dexamethasone inhibited both hypoxia-induced expression/secretion of IL-1β and the ability of hypoxic ACM to induce inflammatory phenotype in HCECs. The ability of hypoxic ACM to up-regulate inflammatory genes in HCECs was inhibited in the presence of IL-1 receptor antagonist (IL-1Ra) and by pretreating ACM with the blocking anti-IL-1β antibody. These findings strongly implicate IL-1β secreted by hypoxic astrocytes in triggering inflammatory activation of HCECs and thereby influencing inflammatory responses at the site of the blood-brain barrier.

Increased expression of bioactive chemokines in human cerebromicrovascular endothelial cells and astrocytes subjected to simulated ischemia in vitro

Leukocyte infiltration into the brain has been implicated in the development of ischemic brain damage. In this study, simulated in vitro ischemia/reperfusion and IL-1beta were found to up-regulate both the expression of intercellular adhesion molecule- (ICAM-1) in cultured human cerebromicrovascular endothelial cells (HCEC) and the adhesion of allogenic neutrophils to HCEC. Both HCEC and human fetal astrocytes (FHAS) also responded to IL-1beta and to in vitro ischemia/reperfusion by a pronounced up-regulation of IL-8 and MCP-1 mRNA and by increased release of IL-8 and MCP-1 in cell culture media. FHAS were found to release 30-times higher levels of MCP-1 than HCEC under both basal and ischemic conditions. However, 100 u/ml IL-1beta induced greater stimulation of both IL-8 and MCP-1 secretion in HCEC (50 and 20 times above controls, respectively) than in FHAS (three and two times above controls, respectively). IL-8 was the principal neutrophil chemoattractant released from IL-1beta-treated HCEC, since IL-8 antibody completely inhibited neutrophil chemotaxis enticed by HCEC media. However, the IL-8 antibody neutralized only 50% of IL-1beta-stimulated neutrophil chemoattractants released from FHAS, and 40%-60% of ischemia-stimulated chemotactic activity released by either HCEC or FHAS. These results suggest that simulated in vitro ischemia, in addition to IL-8 and MCP-1, stimulates secretion of other bioactive chemokines from HCEC and FHAS.

ABC transporters and drug efflux at the blood-brain barrier.

The blood-brain barrier (BBB) is a dynamic physical and biological barrier between blood circulation and the central nervous system (CNS). This unique feature of the BBB lies in the structure of the neurovascular unit and its cerebral micro-vascular endothelial cells. The BBB restricts the passage of blood-borne drugs, neurotoxic substances and peripheral immune cells from entering the brain, while selectively facilitating the transport of nutrients across the BBB into the brain. Thus, the integrity and proper function of the BBB is crucial to homeostasis and physiological function of the CNS. A number of transport and carrier systems are expressed and polarized on the luminal or abluminal surface of the BBB to realize these discrete functions. Among these systems, ABC transporters play a critical role in keeping drugs and neurotoxic substances from entering the brain and in transporting toxic metabolites out of the brain. A number of studies have demonstrated that ABCB1 and ABCG2 are critical to drug efflux at the BBB and that ABCC1 is essential for the blood-cerebral spinal fluid (CSF) barrier. The presence of these efflux ABC transporters also creates a major obstacle for drug delivery into the brain. We have comprehensively reviewed the literature on ABC transporters and drug efflux at the BBB. Understanding the molecular mechanisms of these transporters is important in the development of new drugs and new strategies for drug delivery into the brain.

Development of rapid staining protocols for laser-capture microdissection of brain vessels from human and rat coupled to gene expression analyses

Laser-capture microdissection (LCM) is a technique that enables selective extraction of desired cells from heterogeneous tissues compatible with subsequent molecular analyses. The specific visualization of desired cell types prior to LCM is essential for achieving selective capture. We have developed rapid and selective staining protocols for LCM extraction of microvessels from human and rat brain. Vessels in human and rat brain sections were visualized by a 2 min exposure to fluorescein-labeled lectins Ulex Europeaus Agglutinin I (UEA I) and Ricinus Communis Agglutinin I (RCA I), respectively. Immunohistochemical staining for the endothelial-specific marker, Factor VIII-related antigen (FVIII-rAg), co-localized with that for either UEA I or RCA I, confirming the selective staining of vascular structures with these lectins. Both brain vessels and perivascular parenchyma were captured using LCM, followed by RNA isolation. RT-PCR analyses demonstrated the enrichment of LCM-captured vessels and parenchyma in FVIII-rAg and GFAP mRNA, respectively. LCM-captured human vessels also expressed the tight junction-specific gene, zonula occludens 1 (ZO-1). LCM extraction of vessels from brain sections can be used to perform molecular fingerprinting of neurovascular unit in various brain pathologies.

Biochemical and behavioral characterization of the double transgenic mouse model (APPswe/PS1dE9) of Alzheimer’s disease

ObjectiveThe double transgenic mouse model (APPswe/PS1dE9) of Alzheimer’s disease (AD) has been widely used in experimental studies. β-Amyloid (Aβ) peptide is excessively produced in AD mouse brain, which affects synaptic function and the development of central nervous system. However, little has been reported on characterization of this model. The present study aimed to characterize this mouse AD model and its wild-type counterparts by biochemical and functional approaches.MethodsBlood samples were collected from the transgenic and the wild-type mice, and radial arm water maze behavioral test was conducted at the ages of 6 and 12 months. The mice were sacrificed at 12-month age. One hemisphere of the brain was frozen-sectioned for immunohistochemistry and the other hemisphere was dissected into 7 regions. The levels of Aβ1–40, Aβ1–42 and 8-hydroxydeoxyguanosine (8-OHdG) in blood or/and brain samples were analyzed by ELISA. Secretase activities in brain regions were analyzed by in vitro assays.ResultsThe pre-mature death rate of transgenic mice was approximately 35% before 6-month age, and high levels of Aβ1–40 and Aβ1–42 were detected in these dead mice brains with a ratio of 1:10. The level of blood-borne Aβ at 6-month age was similar with that at 12-month age. Besides, Aβ1–40 level in the blood was significantly higher than Aβ1–42 level at the ages of 6 and 12 months (ratio 2.37:1). In contrast, the level of Aβ1–42 in the brain (160.6 ng/mg protein) was higher than that of Aβ1–40 (74 ng/mg protein) (ratio 2.17:1). In addition, the levels of Aβ1–40 and Aβ1–42 varied markedly among different brain regions. Aβ1–42 level was significantly higher than Aβ1–40 level in cerebellum, frontal and posterior cortex, and hippocampus. Secretase activity assays did not reveal major differences among different brain regions or between wild-type and transgenic mice, suggesting that the transgene PS1 did not lead to higher γ-secretase activity but was more efficient in producing Aβ1–42 peptides. 8-OHdG, the biomarker of DNA oxidative damage, showed a trend of increase in the blood of transgenic mice, but with no significant difference, as compared with the wild-type mice. Behavioral tests showed that transgenic mice had significant memory deficits at 6-month age compared to wild-type controls, and the deficits were exacerbated at 12-month age with more errors.ConclusionThese results suggest that this mouse model mimics the early-onset human AD and may represent full-blown disease at as early as 6-month age for experimental studies.摘要目的阿尔茨海默病(Alzheimer’s disease, AD) APPswe/PS1dE9双转基因小鼠已被广泛运用于各种实验研究。 AD小鼠脑内产生过量的β淀粉样蛋白(Aβ), 后者会影响突触功能和中枢神经系统的发育。 然而, 该转基因小鼠模型的生化和行为学特征却未见报道。 本研究旨在对该小鼠模型的病理从生化和行为学角度进行检测。方法对6月和12月龄转基因和野生型小鼠取血约100 μL, 1 200 g离心后, 分离血清。 在小鼠6月和12月龄时, 进行为期15天的辐射状六臂水迷宫实验。 ELISA法检测血清和大脑中Aβ1–40和Aβ1–42的含量, 以及血清中8-羟基脱氧鸟苷的含量。 比较转基因和野生型小鼠大脑不同部位中α-, β- 和 γ-分泌酶活性的差异。结果在6月龄之前, APPswe/PS1dE9双转基因小鼠的死亡率约为35%, 这些死亡的小鼠脑内Aβ1–40和Aβ1–42水平较高, 两者比例约为1:10。 在6月和12月龄时, 转基因小鼠血清中Aβ1–40水平均显著高于Aβ1–42水平, Aβ1–40与Aβ1–42比例为 2.37:1。 在12月龄时, 转基因小鼠大脑中Aβ1–42水平显著高于Aβ1–40水平, 两者比例约为2.17: 1, 并且在不同脑区中, Aβ1–42和Aβ1–40含量变化较大。 在小脑、 前、 后部皮质层以及海马中, Aβ1–42水平显著高于Aβ1–40。 分泌酶活性在转基因和野生型小鼠之间以及在不同脑部位之间没有很大的差异, 这提示PS1转基因并没有导致高γ-分泌酶活性, 该基因可能使 γ-分泌酶更有效的切割和产生Aβ1–42。 此外, 转基因小鼠血清中8-羟基脱氧鸟苷含量较野生型小鼠升高, 但没有显著性差异。 行为学结果显示, 在6月龄时, 转基因小鼠与野生型相比呈现出显著的记忆障碍, 到12月龄时, 这种障碍变得更为严重, 表现为水迷宫实验中产生更多的错误。结论APPswe/PS1dE9双转基因小鼠最早在6月龄时就能很好地模拟早发性AD, 可用于实验研究。